IL-1 Genes in Disease
The IL-1gene cluster is located on the long arm of human chromosome 2 (2q13) and contains at least the genes for IL-1xcex1 (IL1A), IL-1xcex2 (IL1B), and the IL-1 receptor antagonist (IL-1RN) within a region of 430 Kb (Nicklin, et al., Genomics 19:382-4 (1994)). The agonist molecules, IL-1xcex1 and IL-1xcex2, have potent pro-inflammatory activity and initiate many inflammatory cascades. The IL-1xcex1 and IL-1xcex2 proteins are less than 30 per cent homologous to each other, but both species bind to the same cell surface receptors and their biological activities as cytokines appear to be similar. IL-1 cytokine activity is antagonized by two naturally-occurring inhibitorsxe2x80x94the IL-1 receptor antagonist, and the IL-1 type II receptor. The IL-1 receptor antagonist is structurally homologous to IL-1xcex1 and IL-1xcex2 and binds to IL-1 type I receptors but is biologically inactive, so that it functions as a competitive inhibitor of IL-1. In contrast, the interleukin-1 type II receptor antagonizes IL-1 action by competitively inhibiting IL-1 binding to the type I receptor. In certain cell types, IL-1 receptor antagonist mRNA is spliced to remove the signal sequence so it is not secreted, but the function of intracellular IL-1 receptor antagonist is not known.
The IL-1 receptor antagonist and IL-1 type II receptors appear to play important roles as naturally-occurring antagonists of IL-1 function. Inappropriate production of IL-1 appears to play a central role in the pathology of many autoimmune and inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disorder, Type I diabetes, and psoriasis. IL-1RN allele 2 is associated with osteoporosis (U.S. Pat. No. 5,698,399), nephropathy in diabetes mellitus (Blakemore, et al., Hum. Genet., 97:369-74 (1996)), alopecia areata (Cork, et al., J. Invest. Dermatol., 104(5 Supp.): 15S-16S (1995)), Graves disease (Blakemore, et al., J. Clin. Endocrinol., 80(1): 111-5 (1995)), systemic lupus erythematosus (Blakemore, et al., Arthritis Rheum., 37:1380-85 (1994)), lichen sclerosis (Clay, et al., Hum. Genet., 94:407-10 (1994)), and ulcerative colitis (Mansfield, et al., Gastoenterol., 106(3):637-42 (1994)).
Furthermore, still other diseases and conditions have been associated with IL-1 fimction through the identification of specific human polymorphisms which are associated with an increased risk for developing these diseases and conditions. For example, coronary artery disease (PCT/US98/04725), is associated with IL-1B (-511) allele 2 and IL-1RN (VNTR) allele 2. Diabetic retinopathy (PCT/GB97/02790) is associated with IL1-RN (VNTR). Low birth weight, pregnancy complications and severe periodontal disease (U.S. Pat. No. 5,686,246) are associated with IL-1A (-889) allele 2 and IL-1B (3954) allele 2. The IL-1B (3954) allele 2 is associated with psoriasis and insulin dependent diabetes in DR3/4 patients (di Giovine, et al., Cytokine 7: 606 (1995); Pociot, et al., Eur J. Clin. Invest. 22: 396-402 (1992)). In addition, there are stable inter-individual differences in the rates of production of IL-1, and some of this variation may be accounted for by genetic differences at IL-1 gene loci (Molvig, et al., Scand J. Immunol., 27:705-16 (1988); Pociot, et al., Eur. J. Clin. Invest., 22:396-402 (1992)). Furthermore allele 2 of IL-1RN (VNTR) is associated with ulcerative colitis in Caucasian populations from North America and Europe (Mansfield, J. et al., (1994) Gastroenterology 106: 637-42), particularly within populations of ethnically related Ashkenazi Jews (PCT WO97/25445). Thus, the IL-1 genes are likely mediators of many inflammatory diseases.
Inflammation is now generally regarded as an important component of the pathogenic process of atherosclerosis (Munro, Lab Invest., 58:249-261 (1988), Badimon, et al., Circulation, 87:3-16 (1993), Liuzzo, et al., N.E.J.M., 331(7):417-24 (1994), Alexander, N.E.J.M., 331(7):468-9 (1994)). Several inflammatory products, including IL-1xcex2, have been identified in atherosclerotic lesions or the endothelium of diseased coronary arteries (Galea, et al., Ath. Thromb. Vasc. Biol., 16:1000-6 (1996)). Also, serum concentrations of IL-1xcex2 are elevated in patients with coronary disease (Hasdai, et al., Heart, 76: 24-8 (1996)). Although it was historically believed that the presence of inflammatory agents was responsive to injury or monocyte activation, it is now thought that an abnormal inflammatory response may be causative of coronary artery disease or create an increased susceptibility to the disease. Indeed, particular IL-1RN and IL-1B alleles have been found to be overrepresented in patients with CAD (PCT/US98/04725).
Transgenic Animal Models
There are two basic types of animals with genetically manipulated genomes. A traditional transgenic mammal has a modified gene introduced into its genome and the modified gene can be of exogenous or endogenous origin. A xe2x80x9cknock-outxe2x80x9d mammal is a special type of transgenic mammal, characterized by suppression of the expression of an endogenous gene through genetic manipulation. The disruption of specific endogenous genes can be accomplished by deleting some portion of the gene or replacing it with other sequences to generate a null allele. Cross-breeding mammals having the null allele generates a homozygous mammals lacking an active copy of the gene.
A number of such mammals have been developed, and are extremely helpful in medical development. For example, U.S. Pat. No. 4,736,866 describes a mouse containing a transgene encoding an oncogene. U.S. Pat. No. 5,175,383 describes a mouse with a transgene encoding a gene in the int-2/FGF family. U.S. Pat. No. 5,616,491 describes knock-out mice having suppression of CD28 and CD45. Furthermore, U.S. Pat. No. 5,824,837 describes a transgenic mouse that expresses a human IL-1B transgene. This transgenic mouse is useful for studying specific inflammatory processes mediated by IL-1. Similarly WO 9723129 describes a transgenic animal expressing IL-1a which can be used in research and development of remedies for inflammatory processes mediated by IL-1a.
Transgenic animal models of IL-1 mediated inflammatory diseases would be very useful for identifying pharmaceutical agents that are able to treat or prevent inflammatory diseases.
In one aspect, the invention provides transgenic non-human organisms and cell lines for use in the in vivo screening and evaluation of drugs or other therapeutic regimens useful in the treatment of inflammatory disorders. In one embodiment, the invention is a transgenic animal with a targeted disruption in an interleukin-1 gene. In particular, the gene is the IL-1RN gene. The animal may be chimeric, heterozygotic or homozygotic for the disrupted gene. Homozygotic knock-out IL-1RN mammals have a strong tendency towards developing an inflammatory condition, such as rheumatoid arthritis, inflammatory bowel disorder, Type I diabetes, psoriasis, osteoporosis, nephropathy in diabetes mellitus, alopecia areata, Graves disease, systemic lupus erythematosus, lichen sclerosis, ulcerative colitis, coronary artery disease, arteritic disorders, diabetic retinopathy, low birth weight, pregnancy complications, severe periodontal disease, psoriasis and insulin dependent diabetes, but is particularly characterized by arteritic lesions. The targeted disruption may be anywhere in the gene, subject only to the requirement that it inhibit production of functional IL-1ra protein. In a preferred embodiment, the disruption occurs from the EcoRV site in exon 3 to the first XbaI site in exon 4 of the wild type gene. The transgenic animal may be of any species (except human), but is preferably a mammal. In a preferred embodiment, the non-human animal comprising a targeted disruption in the interleukin-1 receptor antagonist gene, wherein said targeted disruption inhibits production of wild-type interleukin-1 receptor antagonist so that the phenotype of a non-human mammal homozygous for the targeted disruption is characterized by an inflammatory condition.
In another aspect, the invention features a cell or cell line, which contains a targeted disruption in the interleukin-1 receptor antagonist gene. In a preferred embodiment, the cell or cell line is an undifferentiated cell, for example, a stem cell, embryonic stem cell, oocyte or embryonic cell.
Yet in a further aspect, the invention features a method of producing a non-human mammal with a targeted disruption in an interleukin-1 gene. For example, an IL-1RN knock-out construct can be created with a portion of the IL-1RN gene having an internal portion of said IL-1RN gene replaced by a marker. The knock-out construct can then be transfected into a population of embryonic stem m(ES) cells. Transfected cells can then be selected as expressing the marker. The transfected ES cells can then be introduced into an embryo of an ancestor of said mammal. The embryo can be allowed to develop to term to produce a chimeric mammal with the knock-out construct in its germline. Breeding said chimeric mammal will produce a heterozygous mammal with a targeted disruption in the IL-1RN gene. Homozygotes can be generated by crossing heterozygotes.
In another aspect, the invention features IL-1RN knock-out constructs, which can be used to generate the animals described above. In one embodiment, the IL-1RN construct can comprise a portion of the interleukin-1 receptor antagonist (IL-1RN) gene, wherein an internal portion of said IN-1RN gene is replaced by a selectable marker. Preferably, the marker is the neo gene and the portion of the IL-1RN gene is at least 2.5 kb long or 7.0 or 9.5 kb long (including the replaced portion and any IL-1RN flanking sequences). The internal portion preferably covers at least a portion of an exon and most preferably it is from the EcoRV site of exon 3 to the XbaI site of exon 4.
In still another aspect, the invention features methods for testing agents for effectiveness in treating and/or preventing an inflammatory condition. In one embodiment, the method can employ the transgenic animal or cell lines, as described above. For example, a test agent can be administered to the transgenic animal and the ability of the agent to ameliorate the inflammatory condition can be scored as having effectiveness against said inflammatory condition. Any inflammatory condition with an IL-1 component can be tested using these mammals, but in particular, conditions characterized by arteritic lesions are studied. The method may also be used to test agents that are effective against the IL-1 inflammatory proteins and their downstream components.